Genomic Characterization and Antimicrobial Resistance Profile of Streptococcus uberis Strains Isolated from Cows with Mastitis from Northwestern Spain

<b>Background/Objectives</b>: <i>Streptococcus uberis</i> is a Gram-positive bacterium and a major cause of bovine mastitis. The use of antimicrobial treatments raises concerns about resistance. This study aimed to characterize <i>S. uberis</i> isolates from one of the ten largest milk-producing regions in Europe. <b>Methods</b>: Thirty-six isolates from 36 cows with mastitis were identified using MALDI-TOF and VITEK^®MS. Susceptibility to 9 antibiotics (penicillin G, ampicillin, tetracycline, erythromycin, clindamycin, cefotaxime, ceftriaxone, levofloxacin, and moxifloxacin) was determined with VITEK^®2. Whole-genome sequencing was performed using MinION Mk1C. <b>Results</b>: Alleles were identified for 7 loci: <i>arcC</i>, <i>ddl</i>, <i>gki</i>, <i>recP</i>, <i>tdk</i>, <i>tpi</i>, and <i>yqiL</i>. Only 10 isolates had alleles for all the loci. The loci with the highest number of alleles were <i>ddl</i> and <i>tdk</i> (33/36 strains), while <i>arcC</i> had the fewest (19/36). Four isolates were assigned to known sequence types (ST6, ST307, and ST184), and novel alleles were detected in 32 of the 36 isolates. Twelve isolates showed phenotypic resistance to one or more of the following antibiotics: tetracycline, erythromycin, clindamycin, and ceftriaxone. The <i>lnu</i> was the most frequently detected resistance gene (27 out of 102 total gene appearances). A total of 19 virulence factors were identified. All strains were predicted to be capable of infecting human hosts. <b>Conclusions</b>: <i>Streptococcus uberis</i> is a potential reservoir of antimicrobial resistance genes. The use of antimicrobials to treat bovine mastitis has reduced the susceptibility of this microorganism to several antibiotics, underscoring the importance of monitoring antimicrobial use in veterinary practice. The results also highlight the high genetic diversity of the isolates, suggesting a strong capacity to adapt to different environmental conditions.